CRYPTOCHROMES promote daily protein homeostasis

Abstract

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K + abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRYdeficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Circadian regulation of macromolecular complex turnover and proteome renewal

“…Consistent with similar previous studies, <10% of detected proteins showed any significant variation over the circadian cycle (Fig 2E). This is also expected considering the long average half-life (∼days) of mammalian proteins (Mathieson et al , 2018; Schwanhäusser et al , 2011; Wong et al , 2022). Amongst those with significant temporal variation, we found that similar proportions of the proteome showed rhythms in synthesis as rhythms in abundance (Fig 2E).…”

“…Over two days under constant conditions we observed a significant ∼24h oscillation in proteasomal trypsin-like and chymotrypsin-like activities of the proteasome, but not caspase-like activity (Fig 1C). Moreover, we detected a significant interaction between genotype and biological time when comparing trypsin-like proteasome activity between wild type and Cryptochrome1/2-deficient cells, that lack canonical circadian transcriptional feedback repression (Fig S1B, (Wong et al , 2022)). Previous proteomics studies under similar conditions have revealed minimal circadian variation in proteasome subunit abundance (Wong et al , 2022), suggesting that proteasome activity rhythmicity, and therefore rhythms in UPS-mediated protein degradation, are regulated post-translationally (Marshall & Vierstra, 2019; Hansen et al , 2021).…”

“…Finally, given the extensive links between proteome imbalance and many pathological states, daily regulation of protein metabolism has implications for health and disease. Circadian disruption is already strongly associated with impaired proteostasis, though causal mechanisms are poorly understood at this time (Bolitho et al , 2014; Musiek et al , 2018; Leng et al , 2019; Lipton et al , 2017; Wong et al , 2022). In this study we predicted and validated that daily turnover rhythms confer daily variation on the sensitivity of cells and tissues to a clinically relevant proteasome inhibitor.…”

Circadian rhythms in immunity and host‐parasite interactions

“…Recently, multi‐omic analyses have demonstrated a weak overlap between rhythmic gene transcripts and their encoded proteins, challenging the widely accepted view that timing instructions proceed linearly from transcription to translation 19,20 . Interestingly, core clock proteins have been shown to act post‐translationally to regulate protein synthesis and improve robustness of circadian outputs 21,22 …”

“…19,20 Interestingly, core clock proteins have been shown to act post-translationally to regulate protein synthesis and improve robustness of circadian outputs. 21,22 Whilst the search continues for a communicating factor between classic TTFLs and non-TTFL mechanisms of circadian regulation, these data provide insight to the layers of complexity within circadian systems and encourages researchers to consider non-TTFL mechanisms driving circadian observations, especially in cases where canonical clock gene homologues have yet to be identified, such as in most parasites.…”